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1.
Experiments examined the metabolic basis of Ca2+-induced conidiation during the 12-h period following the addition of Ca2+ to 40-h vegetative cultures ofPenicillium notatum. Vegetative mycelium had enzymic capacity for three routes of glucose catabolism viz. the Embden-Meyerhof-Parnas (EMP), pentose phosphate (PP) and the Entner-Doudoroff (ED) sequences. Inhibitors of EMP enzymes restricted vegetative growth more than that associated with conidiation whilst arsenate augmented the limited capacity of lower levels of Ca2+ to promote conidiation. Arsenite (5.6 mmol · 1–1) partially blocked the metabolism of pyruvate and caused its accumulation, which was also promoted by Ca2+ alone. Arsenite did not induce conidiation in vegetative cultures but when combined with Ca2+ it enhanced conidiation. Radiorespirometry and the analysis of accumulated pyruvate, promoted by arsenite, indicated that approximately 54% of carbon was catabolized via combined EMP/ED routes and 46% by the PP pathway and subsequently via a weakly functional TCA cycle. Calcium-induced cultures swung to a primarily ED (25%) and PP (75%) based catabolism with low substrate level phosphorylation, including a facility for a non-phosphorylative ED route, and further diminished oxidative TCA capacity. Pyruvate accumulation in Ca2+-induced cultures coincided with the decline in activity of pyruvate dehydrogenase and a reduced capacity for gluconeogenesis, with other enzymes of pyruvate metabolism showing altered activities. These changes in enzyme activities, pyruvate accumulation and its subsequent metabolism were related to growth rate and the developmental cycle, and are discussed in conjunction with the regulatory role of calcium.  相似文献   

2.
Encystment induction of Colpoda cucullus is promoted by an increase in external Ca2+ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca2+ or without the addition of Ca2+. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca2+/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca2+ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca2+-triggered signaling pathways, may be involved in encystment induction.  相似文献   

3.
The TCA cycle was examined during Ca2+-induced conidiation in Penicillium notatum over the 12-h period after addition of Ca2+ to vegetative cultures. Conidiation was independent of Ca2+ when certain intermediates and derivatives of the TCA cycle served as sole carbon sources. Arsenite and malonate augmented the effect of Ca2+ on conidiation but did not substitute for it. Mitochondria from vegetative cells had low rates of oxidation of TCA cycle intermediates and, with the exception of pyruvate, aconitate and glutamate, these were poorly linked to phosphorylation processes. Calcium ions affected mitochondrial function causing reduced oxidation of oxoglutarate, elimination of pyruvate oxidation and a decline in respiratory control of these substrates with increased oxidation of NADH and NADPH. Radiorespirometric studies and enzyme searches revealed a complete but weakly oxidative TCA cycle in vegetative cells. In Ca2+-induced cells oxoglutarate dehydrogenase activity was deleted within 6.5 h of Ca2+ addition and this was accompanied by establishment of an incomplete Krebs cycle. Calcium-induced conidiation was associated with increased capacity for acetate and glutamate metabolism involving an activated glyoxylate shunt which may be related to enhanced biosynthetic demand. The metabolic basis of the Ca2+ effect on conidiation is discussed in connection with previous findings.  相似文献   

4.
Summary A fungal elicitor extracted fromAspergillus oryzae (Ahlb.) Cobn mycelia promoted the production of shikonin derivatives inOnosma paniculatum Bur et Franch cell suspension cultures. Elicitor treatment also increased Ca2+ concentration in RM9 medium, which could be measured earlier than the elicited increase of shikonin formation. Several reagents known to induce Ca2+-influx and increase the intracellular-free Ca2+ level, such as the addition of Ca (NO3)2·4H2O, the Ca2+ ionophore A23187, and abscisic acid (ABA), appreciably suppressed the elicitor-promoted shikonin formation inOnosma cells. In contrast, the decrease of intracellular-free Ca2+ level by the specific Ca2+-chelator ethylene glycol bis (β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) or the Ca2+—channel blocker, verapamil, enhanced the biosynthesis of shikonin even in the absence of elicitor. Treatment of cells with trifluoperazine (TFP) also stimulated shikonin formation inOnosma cell cultures. A rapid and transient drop of free Ca2+ level in one protoplast was directly determined after the addition of elicitor toOnosma cell cultures. The inhibitory effect on shikonin formation by ABA was largely on account of its ability to restore the intracellular Ca2+ level lowered by the elicitor. These results suggest that Ca2+ play a significant role in an early stage of the elicitation process ofOnosma cells. The rapid drop of cytoplasmic Ca2+ carries the elicitor signal and in turn regulates the biosynthesis of shikonin derivatives.  相似文献   

5.
Stimulation of Ehrlich ascites tumor cells with leukotriene D4 (LTD4) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca2+ concentration, [Ca2+] i . The Ca2+ peak time, i.e., the time between addition of LTD4 and the highest measured [Ca2+] i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD4 (100 nm), the highest measured increase in [Ca2+] i in Ehrlich cells suspended in Ca2+-containing medium is 260 ± 14 nm and the EC50 value for LTD4-induced Ca2+ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC4 and LTE4 nor LTB4 are able to mimic or block the LTD4-induced Ca2+ mobilization, hence the receptor is specific for LTD4. Removal of Ca2+ from the experimental buffer significantly reduces the size of the LTD4-induced increase in [Ca2+] i . Furthermore, depletion of the intracellular Ins(1,4,5)P3-sensitive Ca2+ stores by addition of the ER-Ca2+-ATPase inhibitor thapsigargin also reduces the size of the LTD4-induced increase in [Ca2+] i in Ehrlich cells suspended in Ca2+-containing medium, and completely abolishes the LTD4-induced increase in [Ca2+] i in Ehrlich cells suspended in Ca2+-free medium containing EGTA. Thus, the LTD4-induced increase in [Ca2+] i in Ehrlich cells involves an influx of Ca2+ from the extracellular compartment as well as a release of Ca2+ from intracellular Ins(1,4,5)P3-sensitive stores. The Ca2+ peak times for the LTD4-induced Ca2+ influx and for the LTD4-induced Ca2+ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD4 also induces a transient increase in Ins(1,4,5)P3 generation in the Ehrlich cells, and the Ins(1,4,5)P3 peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P3 content seems to increase before the LTD4-induced Ca2+ release from the intracellular stores but after the LTD4-induced Ca2+ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD4-induced increase in Ins(1,4,5)P3 as well as the LTD4-induced increase in [Ca2+] i , indicating that a U73122-sensitive phospholipase C is involved in the LTD4-induced Ca2+ mobilization in Ehrlich cells. The LTD4-induced Ca2+ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD4-activated Ca2+ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD4 acts in the Ehrlich cells via a specific receptor for LTD4, which upon stimulation initiates an influx of Ca2+, through yet unidentified Ca2+ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P3 formation and finally release of Ca2+ from the intracellular Ins(1,4,5)P3-sensitive stores. Received: 9 February 1996/Revised: 15 August 1996  相似文献   

6.
Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca2+]e) with increases in intracellular calcium concentration ([Ca2+]i) that include single-cell Ca2+ oscillations and propagated intercellular Ca2+ waves. The rate and pattern of propagation of low [Ca2+]e-induced intercellular Ca2+ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca2+ wave propagation. The low [Ca2+]e-induced Ca2+ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca2+]e-induced response is also blocked by replacement of extracellular Ca2+ with Ba2+, Zn2+, or Ni2+, and by 100 µM La3+. Glial cells in lowered [Ca2+]e(0.1–0.5 mM) show an increased [Ca2+]i response to bath application of ATP, whereas glial cells in increased [Ca2+]e (10–15 mM) show a decreased [Ca2+]i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca2+]e and the release of Ca2+ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca2+ such as seizures or ischemia.  相似文献   

7.
The rate of the45Ca2+ uptake by the submergedTrichoderma viride mycelium increased with the age of the culture from 6 h until a maximum which was reached at about 30 h, and then decreased until the uptake was virtually zero. The decrease in the rate of the45Ca2+ uptake was accompanied by an increase of mycelial mass. The uptake rate could not be reactivated upon substituting the medium for a fresh one, without or with dilution of the mycelium. The results suggest that the rate of45Ca2+ uptake reflects the biological age of the submerged culture. The surface-cultivated mycelium took up45Ca2+ proportionally with time. The autoradiography of colonies showed that45Ca2+ was distributed homogeneously throughout the mycelium during vegetative growth while conidiation was accompanied by a massive accumulation of45Ca2+ in conidia. This work was supported by theSwiss National Science Foundation (joint Swiss-Slovak project 75LPJ041485) andSlovak Grant Agency (grant no. 1/1158/93).  相似文献   

8.
Abstract Using the method of compartmental analysis, the ion fluxes and compartment concentrations of Ca2+, K+ and Cl- have been compared in the untreated vegetative frond and the abscisic acid (ABA) induced turion of Spirodela polyrrhiza. The ABA-induced turion is characterized by reduced Ca2+ exchange across the tonoplast and low vacuolar Ca2+ concentration relative to the vegetative frond. In addition the turion exhibits a higher plasmalemma flux with a correspondingly high Ca2+ concentration in the cytoplasm. The concentration of K+ and Cl- is much lower in the cytoplasm of the ABA-induced turion than in the vegetative frond with the influx/efflux ratio at both the plasmalemma and the tonoplast being less than 1, a finding exhibited also in dormant storage tissue. Treatment of vegetative fronds with ABA for 18 h resulted in a reduced K+ plasmalemma efflux relative to untreated vegetative fronds and a concomitant increase in the cytoplasmic concentration. There was no rapid effect of ABA on Ca2+, K+ or Cl- fluxes through either membrane. These results are consistent with the notion that drastic changes in ion fluxes and concentrations in the turion are a secondary consequence of ABA-induced development, possibly due to prior regulation by ABA of enzymes inherent to processes involved in membrane transport.  相似文献   

9.
The invasion-associated type III secretion system (T3SS-1) of S. Typhimurium is required to initiate and sustain an acute inflammatory response in the intestine. We investigated the relationship of S. Typhimurium T3SS-1-induced IL-8 expression and invasion with intracellular Ca2+ mobilization in HeLa cells. Compared to the sipAsopABDE2 mutant, strains carrying a mutation in sipA, or mutations in sopABDE2 induced higher levels of IL-8 and greater bacterial internalization despite the fact that these mutants elicited similarly low intracellular concentrations of Ca2+. Likewise, complemented sipAsopABDE2 mutant with sopE2 did not affect intracellular Ca2+ concentrations or IL-8 expression, but significantly increased bacterial internalization. Treating HeLa cells with the calcium chelator BAPTA-AM or with D-BAPTA-AM, a derivative with greatly reduced Ca2+ chelating activity, yielded strong evidence that BAPTA-AM does not affect invasion and inhibits IL-8 secretion by a calcium-dependent mechanism. These findings suggest that, although wild-type S. Typhimurium-induced IL-8 expression and bacterial internalization in HeLa cells coincides with increased cytosolic Ca2+, the differing levels of IL-8 and invasion induced by strains carrying different effector proteins are unrelated to levels of intracellular Ca2+.  相似文献   

10.
11.
1-Methyl-4-phenylpyridinium (MPP+) or 6-hydroxydopamine (6-OHDA) caused a nuclear damage, the mitochondrial membrane permeability changes, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species and the depletion of GSH in PC12 cells. Nicardipine (a calcium channel blocker), EGTA (an extracellular calcium chelator), BAPTA-AM (a cell permeable calcium chelator) and calmodulin antagonists (W-7 and calmidazolium) attenuated the MPP+-induced mitochondrial damage and cell death. In contrast, the compounds did not reduce the toxicity of 6-OHDA. Treatment with MPP+ or 6-OHDA evoked the elevation of intracellular Ca2+ levels. Unlike cell injury, addition of nicardipine, BAPTA-AM and calmodulin antagonists prevented the elevation of intracellular Ca2+ levels due to both toxins. The results show that the MPP+-induced formation of the mitochondrial permeability transition seems to be mediated by elevation of intracellular Ca2+ levels and calmodulin action. In contrast, the 6-OHDA-induced cell death seems to be mediated by Ca2+-independent manner.  相似文献   

12.
Summary A theory for Na+, K+ and Ca2+ competitive adsorption to a charged membrane is used to explain a number of experimental observations in smooth muscle. Adsorption is described by Langmuir isotherms for mono- and divalent cations which in turn are coupled in a self-consistent way to the bulk solution through the diffuse double layer theory and the Boltzman equations. We found that the dissociation constants for binding of Na+, K+ and Ca2+ in guinea pig taenia coli areca. 0.009, 1.0, and 4×10–8 m, respectively. Furthermore, the effect of a Ca2+ pump that maintains free surface Ca2+ concentration constant is investigated. A decrease in intracellular Na+ content results in an increased Ca2+ uptake; part of this uptake is due to an increase in surface-bound Ca2+ in an intracellular compartment which is in contact with the myofilaments. Variations in the amount of charge available to bind Ca2+ and the surface charge density are studied and their effect interpreted in terms of different pharmacological agents.  相似文献   

13.
The paper describes our concept about the existence of a certain strategy of rearrangements of ionic mechanisms of he intracellular trigger signal transmission in muscles during their contractile function evolution. It is shown that the rearrangements of muscles to accelerate the single (discrete) contraction cycle is accompanied by a change of mechanisms of external stimulus transduction into an intracellular trigger signal: direct activation of intracellular effectors by extracellular Ca2+ is replaced by indirect mechanisms of Ca2+-, then Ca2+- and Na+-induced, and in skeletal muscle fibers of vertebrates (SMFV) of Na+-induced Ca2+ release from the intracellular depot, sarcoplasmic reticulum. These rearrangements promoted an intensification of the Ca2+ intracellular mobilization to provide for the most complete pulse control of SMFV phasic contractions by the CNS and their protection from undesirable peripheral influences.  相似文献   

14.
Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 μM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 μM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.  相似文献   

15.
Cytosolic Ca2+ and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca2+ and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl2 (3, 23, and 43 mM) and MJ (0, 10, and 100 μM) on day 6 of growth. Increased Ca2+ influx through the addition of extracellular CaCl2 suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 μM). In the presence of 3 mM CaCl2 in the medium, the addition of Ca2+ chelator EGTA (1, 2.5, and 5 mM) or Ca2+ channel blocker verapamil (1, 10, and 50 μM) to MJ-induced (100 μM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca2+ inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca2+ concentration and a low extracellular Ca2+ concentration (3 mM) enhanced MJ-induced ajmalicine production.  相似文献   

16.
Summary The voltage- and time-dependent K+ current,I K + out , elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La3+) to the extracellular medium. These results suggested that the influx of external Ca2+ was necessary for K+ current activation. The IC50, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 m at a test potential of +60 mV following a 20-min incubation period.In order to test whether intracellular Ca2+ actuated the K+ current, we altered either the Ca2+ buffering capacity or the free Ca2+ concentration of the intracellular medium (pipette filling solution). By these means,I K + out could be varied over a 10-fold range. Increasing the free Ca2+ concentration from 40 to 400nm also shifted the activation of the K+ current toward more negative potentials. Maintaining cytoplasmic Ca2+ at 500nm with 40nm EGTA resulted in a more rapid activation of the K+ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca2+ on depolarization. Increasing intracellular Ca2+ to 500nm or 1 m also led to inactivation of the K+ current within a few minutes. It is concluded thatI K + out is regulated by cytosolic Ca2+, which is in turn controlled by Ca2+ influx through dihydropyridine-, and phenylalkylamine-sensitive channels.  相似文献   

17.
N-acyl-l-homoserine lactones (AHLs) are quorum sensing (QS) signal molecules that are commonly used in gram-negative bacteria. Recently, it has become evident that AHLs can influence the behavior of plant cells. However, little is known about the mechanism of the plants’ response to these bacterial signals. Calcium ions (Ca2+), ubiquitous intracellular second messengers, play an essential role in numerous signal transduction pathways in plants. In this study, the cytosolic free Ca2+ concentration ([Ca2+]cyt) was measured by a luminometric method in the excised root cells of Arabidopsis plants that were treated with N-butyryl-homoserine lactone (C4-HSL). There was a transient and immediate increase in [Ca2+]cyt levels, and the highest level (0.4 μM), approximately 2-fold higher than the basal level, was observed at the 6th second after the addition of 10 μM C4-HSL. Pretreatments with La3+, verapamil or ethylene glycol tetraacetic acid (EGTA) inhibited the increase in [Ca2+]cyt caused by C4-HSL, whereas it remained unaffected by pretreatment with Li+, indicating that the Ca2+ contributing to the increase in [Ca2+]cyt was mobilized from the extracellular medium via the plasma membrane Ca2+ channels but not from the intracellular Ca2+ stores. Furthermore, electrophysiological approaches showed that the transmembrane Ca2+ current was significantly increased with the addition of C4-HSL. Taken together, our observations suggest that C4-HSL may act as an elicitor from bacteria to plants and that Ca2+ signaling participates in the ability of plant cells to sense the bacterial QS signals.  相似文献   

18.
Ion fluxes across the plasma membrane activated by 1 mM Ce4+, cell apoptosis and taxol biosynthesis in suspension cultures ofTaxus cuspidata were studied. The extracellular pH sharply decreased upon the addition of 1 mM Ce4+, then increased gradually and exceeded the initial pH value over a time period of 12 h. The extracellular Ca2+ concentration decreased within the first 3 h after the addition of Ce4+, then gradually decreased to one third of initial value in control at about 72 h and remained unchanged afterwards. Experiments with an ion channel blocker and a Ca2+-channel blocker indicated that the dynamic changes in extracellular pH and the Ca2+ concentration resulted from the Ce4+-induced activation of H+ uptake and Ca2+ influx across the plasma membranevia ion channels. A pretreatment of the ion channel blocker initiated Ce4+-treated cells to undergo necrosis, and the prior addition of the Ca2+-channel blocker inhibited Ce4+-induced taxol biosynthesis and apoptosis. It is thus inferred that H+ uptake is necessary for cells to survive a Ce4+-caused acidic environment and is one of the mechanisms of Ce4-induced apoptosis. Furthermore, the Ca2+ influx across the plasma membrane mediated both the Ce4+-induced apoptosis and taxol biosynthesis.  相似文献   

19.
Effects of the external Ca2+ concentration on the depolarization-induced transient inward Na+ current responsible for the Na+ spike in the dinoflagellate Noctiluca miliaris were examined. The peak value and the duration of the Na+ current increased when lowering the external Ca2+ concentration. The threshold potential level for activation and the reversal potential level of the current were not affected by the external Ca2+ concentration. The inactivation took place even in a solution containing EGTA with very low (<10–9 M) Ca2+ concentration. Voltage dependency of the inactivation was scarcely affected by the external Ca2+ concentration. It is concluded that inactivation of Na+ channels responsible for the current is dependent on membrane depolarization and that the external Ca2+ modulates the inactivation kinetics. Appearance of a Na+ spike in a solution with reduced Ca2+ concentration is caused by a lowered rate of inactivation of the Na+ channels.  相似文献   

20.
The interactions of divalent cations with the adenosine triphosphatase (ATPase) and para-nitrophenyl phosphatase (pNPPase) activity of the purified dog kidney Na pump and the fluorescence of fluorescein isothiocyanate (FITC)-labeled pump were determined. Sr2+ and Ba2+ did not compete with K+ for ATPase (an extracellular K+ effect). Sr2+ and Ba2+ did compete with Na+ for ATPase (an intracellular Na+ effect) and with K+ for pNPPase (an intracellular K+ effect). These results suggest that Ba2+ or Sr2+ can bind to the intracellular transport site, yet neither Ba2+ nor Sr2+ was able to activate pNPPase activity; we confirmed that Ca2+ and Mn2+ did activate. As another measure of cation binding, we observed that Ca2+ and Mn2+, but not Ba2+, decreased the fluorescence of the FITC-labeled pump; we confirmed that K+ substantially decreased the fluorescence. Interestingly, Ba2+ did shift the K+ dose-response curve. Ethane diamine inhibited Mn2+ stimulation of pNPPase (as well as K+ and Mg2+ stimulation) but did not shift the 50% inhibitory concentration (IC50) for the Mn2+-induced fluorescence change of FITC, though it did shift the IC50 for the K+-induced change. These results suggest that the Mn2+-induced fluorescence change is not due to Mn2+ binding at the transport site. The drawbacks of models in which Mn2+ stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn2+ stimulates by binding to both the catalytic and transport sites are presented. Our results provide new insights into the pNPPase kinetic mechanism as well as how divalent cations interact with the Na pump.  相似文献   

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